Molecular analysis of single B cells from T-cell-rich B-cell lymphoma shows the derivation of the tumor cells from mutating germinal center B cells and exemplifies means by which immunoglobulin genes are modified in germinalcenter B cells

T-cell-rich B-cell lymphoma (TCRBCL) belongs to the group of diffuse large cell lymphomas (DLL). It is characterized by a small number of tumor B cell s among a major population of nonmalignant polyclonal T cells. To identify the developmental stage of the tumor progenitor cells, we micromanipulated the putative neoplastic large CD20(+) cells from TCRBCLs and amplified and sequenced immunoglobulin (Ig) V gene rearrangements from individual cells. In six cases, clonal Ig heavy, as well as light chain, gene rearrangements were amplified from the isolated B cells. All six cases harbored somaticall y mutated V gene rearrangements with an average mutation frequency of 15.5% for heavy (V-H) and 5.9% for light (V-L) chains and intraclonal diversity based on somatic mutation. These findings identify germinal center (GC) B c ells as the precursors of the transformed B cells in TCRBCL. The study also exemplifies various means how Ig gene rearrangements can be modified by GC B cells or their malignant counterparts in TCRBCL: In one case, the tumor precursor may have switched from kappa to lambda light chain expression aft er acquiring a crippling mutation within the initially functional kappa lig ht chain gene. In another case, the tumor cells harbor two in-frame VH gene rearrangements, one of which was rendered nonfunctional by somatic mutatio n. Either the tumor cell precursor entered the GC with two potentially func tional in-frame rearrangements or the second V(H)D(H)J(H) rearrangement occ urred in the GC after the initial in-frame rearrangement was inactivated by somatic mutation. Finally, in each of the six cases, at least one cell con tained two (or more) copies of a clonal Ig gene rearrangement with sequence variations between these copies. The presence of sequence variants for V r egion genes within single B cells has so far not been observed in any other normal or transformed B lymphocyte. Fluorescence in situ hybridization (FI SH) points to a generalized polyploidy of the tumor cells. (C) 1999 by The American Society of Hematology.